Angiotensin II induces matrix metalloproteinase-9 expression via a nuclear factor-kappaB-dependent pathway in vascular smooth muscle cells

Angiotensin II (AngII) is widely recognized as a critical regulator of the development of atherosclerosis. Matrix metalloproteinases (MMPs) are thought to participate in plaque destabilization through degradation of the extracellular matrix. In the present study, we investigated the potential mechanism of AngII-induced MMP-9 expression in vascular smooth muscle cells (VSMC). AngII upregulated the expression of MMP-9 significantly in VSMC obtained from rat aorta. RNAi-mediated knockdown of p65 and losartan, an inhibitor of AngII receptors subtype-1 (AT₁), could abolish AngII-induced MMP-9 expression. In addition, AngII induced the NF-κB binding activity via AT₁ and AT₂ receptors in VSMC, and AngII-induced activation of NF-κB is not associated with significant downregulation of IκB. In summary, this study demonstrates that AngII stimulates NF-κB nuclear translocation in VSMC via AT₁ and AT₂. AngII increases the expression of MMP-9 in VSMC, and AT₁ and NF-κB pathways have an important role in this response.     

Cyclic strain-induced endothelial MMP-2: role in vascular smooth muscle cell migration

Matrix metalloproteinases (MMPs) play a vital role in vasculature response to hemodynamic stimuli via the degradation of extracellular matrix substrates. In this study, we investigated the putative role of cyclic strain-induced endothelial MMP-2 (and MMP-9) expression and release in modulating bovine aortic smooth muscle cell (BASMC) migration in vitro. Equibiaxial cyclic strain of bovine aortic endothelial cells (BAECs) leads to elevation in cellular MMP-2 (and MMP-9) expression, activity, and secretion into conditioned media, events which were time- and force-dependent. Subsequent incubation of BASMCs with conditioned media from chronically strained BAECs (5%, 24 h) significantly reduces BASMC migration (38+/-6%), an inhibitory effect which could be completely reversed by targeted siRNA 'knock-down' of MMP-2 (but not MMP-9) expression and activity in BAECs. Moreover, inhibition of strain-mediated MMP-2 expression in BAECs by protein tyrosine kinase (PTK) blockade with genistein (50 microM) was also found to completely reverse this inhibitory effect on BASMC migration. Finally, direct supplementation of recombinant MMP-2 into the BASMC migration assay was found to have no significant effect on migration. However, the effect on BASMC migration of MMP-2 siRNA transfection in BAECs could be reversed by supplementation of recombinant MMP-2 into BAEC media prior to (and for the duration of) strain. These findings reveal a potentially novel role for strain-induced endothelial MMP-2 in regulating vascular SMC migration.     

NR6A1 couples with cAMP response element binding protein and regulates vascular smooth muscle cell migration

Vascular smooth muscle cell (VSMC) migration is implicated in atherosclerosis and restenosis. Nuclear receptor subfamily 6, group A, member 1 (NR6A1) is involved in regulating embryonic stem cell differentiation, reproduction, neuronal differentiation. Functional cooperation between cAMP response element modulator tau (CREMtau) and NR6A1 can direct gene expression in cells. cAMP response element binding protein (CREB) plays a key role in VSMC migration. In this study, we sought to determine whether CREB involved in NR6A1-modulated VSMC migration. VSMCs treated with platelet-derived growth factor-BB (PDGF-BB) displayed reduced mRNA and protein levels of NR6A1. Adenovirus-mediated expression of NR6A1 (Ad-NR6A1) could inhibit PDGF-BB- and serum-induced VSMC migration. The mRNA and protein expressions of secreted phosphoprotein 1 (SPP1) were down-regulated by NR6A1 overexpression. SPP1 promoter reporter activity was repressed by NR6A1. NR6A1 was found to physically couple with nuclear actin and the large subunit of RNA polymerase II. Furthermore, we showed that CREB interacted with NR6A1 in VSMCs. NR6A1 overexpression repressed cAMP response element (CRE) activity. ChIP assay revealed that NR6A1 bind to SPP1 promoter. Luciferase reporter assay showed that NR6A1 regulated SPP1 promoter activity via a putative CRE site. Adenovirus mediated local NR6A1 gene transfer attenuated stenosis after balloon-induced arterial injury in Sprague–Dawley rats. Taken together, this study provided experimental evidence that NR6A1 modulated SPP1 expression via its binding with CREB protein in VSMCs. We also revealed a NR6A1-CREB-SPP1 axis that serves as a regulatory mechanism for atherosclerosis and restenosis.     

Angiotensin II induces MMP 2 activity via FAK/JNK pathway in human endothelial cells

Matrix metalloproteinases (MMPs) play an important role in the pathogenesis of cardiovascular diseases and are modified in response to a variety of stimuli such as bioactive peptides, cytokines and/or grown factors. In this study, we demonstrated that angiotensin II (Ang II) induces a time- and dose-dependent increase in the activity of metalloproteinase 2 (MMP 2) in human umbilical vein endothelial cells (HUVEC). The effect of Ang II was markedly attenuated in cells pretreated with wortmannin and LY294002, two selective inhibitors of phosphatidylinositol-3-kinase (PI3K), indicating that PI3K plays a key role in regulating MMP 2 activity. Similar results were observed when HUVEC were pretreated with genistein, a non-selective tyrosine kinases inhibitor, or with the specific Src-family tyrosine kinase inhibitor PP2, demonstrating the involvement of protein tyrosine kinases, and particularly Src-family tyrosine kinases on the downstream signaling pathway of Ang II receptors. Furthermore, Ang II-induced MMP 2 activation was markedly blocked by SP600125, a selective c-Jun N-terminal kinase (JNK) inhibitor, or pre-treatment of cells with antisense oligonucleotide to focal adhesion kinase (FAK), indicating that both molecules were important for the activation of MMP 2 by Ang II receptor stimulation. In conclusion, these results suggest that Ang II mediates an increase in MMP 2 activity in macrovascular endothelial cells through signal transduction pathways dependent on PI3K and Src-family tyrosine kinases activation, as well as JNK and FAK phosphorylation.     

Angiotensin II type 1 receptor-associated protein regulates carotid intimal hyperplasia through controlling apoptosis of vascular smooth muscle cells

Intimal hyperplasia is the main cause of restenosis after carotid artery injury, and the underlying mechanism involves the proliferation and migration of vascular smooth muscle cells (VSMCs). Angiotensin II Type 1 Receptor-Associated Protein (ATRAP) has been reported to withstand intimal hyperplasia by inhibiting VSMCs proliferation and migration; however, whether the beneficial effect of ATRAP associates with VSMCs apoptosis remains unclarified. We demonstrated that the adenoviral-mediated overexpression of ATRAP induced VSMC apoptosis, alleviating the balloon injury-induced neointima formation in rats. Under the condition of Angiotensin-II stimulation, ATRAP overexpression induced the apoptosis of rat VSMCs by depressing the PI3K-Akt signaling; whereas up-regulation of Akt by PTEN inhibitor abolished the apoptotic death. Thus, ATRAP regulates carotid intimal hyperplasia through controlling the PI3K-Akt signal-mediated VSMCs apoptosis.     

Striking differences of LDL receptor-related protein 1B expression in mouse and human

The low density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a member of the expanding LDL receptor family, and is closely related to LRP. It was discovered as a putative tumor suppressor, and is frequently inactivated in human malignant tissues. However, the expression pattern of LRP1B in normal human tissues was unclear. In the present study, we analyzed LRP1B expression in normal mouse and human tissues. By using RT-PCR, we found that, while mouse LRP1B expression is mostly restricted to the brain, human LRP1B expression is more widespread with highest expression levels detected in the brain, adrenal gland, salivary gland, and testis. Although mouse LRP1B expresses in the forms of both full-length receptor tail and an alternatively spliced form lacking a 33-amino acid insert, human LRP1B is expressed exclusively in the form of full-length receptor tail. Finally, we found that, unlike mouse LRP1B, human LRP1B is cleaved by furin. Taken together, these data demonstrate that there are striking differences between LRP1B expression in mouse and human tissues. The broader expression pattern of LRP1B in human tissues suggests that this putative tumor suppressor may play roles in several types of human cancer.     

Essential role of the low density lipoprotein receptor-related protein in vascular smooth muscle cell migration

The low density lipoprotein receptor-related protein (LRP) is a multifunctional cell surface receptor highly expressed in human aortic smooth muscle cells. In the present study, we used the short interfering RNA (siRNA) technique to explore the role of LRP in smooth muscle cell migration. We identified an LRP-specific siRNA that selective silences LRP expression in human aortic smooth muscle cells. As a consequence, LRP-mediated ligand degradation was significantly reduced. More important, we found that platelet-derived growth factor-dependent cell migration was inhibited in cells transfected with LRP siRNA. These results demonstrate an important role of LRP in smooth muscle cell migration.     

Parallel regulation of arginine transport and nitric oxide synthesis by angiotensin II in vascular smooth muscle cells role of protein kinase C

Summary Experiments were performed to characterize arginine transport in vascular smooth muscle cells (SMCs) and the effect of angiotensin II (Ang II) on this process. In addition, the role of arginine transport in the cytokineinduced nitric oxide (NO) production was assessed. Arginine transport takes place through Na⁺-independent (60%) and Na⁺-dependent pathways (40%). The Na⁺-independent arginine uptake appears to be mediated by system y⁺ because of its sensitivity to cationic amino acids such as lysine, ornithine and homoarginine. The transport system was relatively insensitive to acidification of the extracellular medium. By contrast, the Na⁺-dependent pathway is consistent with system B^0,+ since it was inhibited by both cationic and neutral amino acids (i.e., glutamine, phenylalanine, and asparagine), and did not accept Li⁺ as a Na⁺ replacement. Treatment of SMCs with 100nM Ang II significantly inhibited the Na⁺-dependent arginine transport without affecting systems y⁺, A, and L. This effect occurred in a dose-dependent manner (IC₅₀ of 8.9 ± 0.9nM) and is mediated by the AT-1 receptor subtype because it was blocked by DUP 753, a non-peptide antagonist of this receptor. The inhibition of system B^0,+ by Ang II is mediated by protein kinase C (PKC) because it was mimicked by phorbol esters (phorbol 12-myristate 13-acetate) and was inhibited by staurosporine. Ang II also inhibited the IL-1 induced nitrite accumulation by SMCs. This action was also inhibited by staurosporine and reproduced with phorbol esters, suggesting a coupling between arginine uptake and NO synthesis through a PKC-dependent mechanism. However, arginine supplementation in the medium (10mM) failed to prevent the inhibitory action of Ang II on NO synthesis. These findings suggest that although Ang II inhibits concomitantly arginine transport and NO synthesis in SMCs, the reduction of NO synthesis is not associated with alterations in the cellular transport of arginine.Abbreviations Arg arginine - Orn ornithine - HmR homoarginine - Lys lysine - Gln glutamine - Asn asparagine - His histidine - Phe phenylalanine - Leu leucine - Cys Cysteine - Ala alanine - Ser serine - Thr threonine - Glu glutamate - mAIB -methyl-aminoisobutyric acid - BCH bicycloaminoheptane     

Hypertrophic response to angiotensin II is mediated by protein kinase D-extracellular signal-regulated kinase 5 pathway in human aortic smooth muscle cells

Angiotensin II plays a critical role in hypertrophy of vascular smooth muscle cells, however, the molecular underpinnings remain unclear. The present study indicated that AT₁/PKC/PKD pathway was able to regulate downstream ERK5, affecting pro-hypertrophic responses to Ang II. Ang II-stimulated phosphorylation of ERK5 in a time- and dose-dependent manner in human aortic smooth muscle cells (HASMCs). The pharmacological inhibitors for AT₁ and PKCs significantly inhibited Ang II-induced ERK5 activation, suggesting the involvement of the AT₁/PKC pathway. In particular, PKD was critical for Ang II-induced ERK5 activation since silencing PKD by siRNA markedly inhibited Ang II-induced ERK5 activation. Consequently, we found that Losartan, Gö 6983 and PKD siRNA significantly attenuated ERK5 activated translocation and hypertrophy of HASMCs by Ang II. Taken together, we demonstrated for the first time that Ang II activates ERK5 via the AT₁/PKC/PKD pathway and revealed a critical role of ERK5 in Ang II-induced HASMCs hypertrophy.     

Cell-cell bond modulates vascular smooth muscle cell responsiveness to Angiotensin II

Cell attachment is provided by cell-matrix and cell-cell bonds, and acts as a regulator of vascular smooth muscle cell (VSMC) survival, activity and homeostasis, as well as of VSMCs response to pathogenic stimuli. In this work we elicited an exclusive cell-cell contact by culturing A7r5 VSMCs on agarose-coated wells to form floating cell clusters, and we demonstrated that a steady state with a reduced response to the vasoactive peptide Angiotensin II (ATII) was induced. We found that clustered VSMCs showed subcortical stabilization of β-catenin and Caveolin 1 (Cav1), unlike adherent confluent counterparts. We demonstrated that β-catenin and Cav1 stabilization at the membrane level hampers the molecular cross-talk induced by ATII-activated AT1 receptor (AT1R), thereby impeding the phosphorylation of Cav1 and IGF1R, the NADPH oxidase activity, and counteracting ATII-dependent hypertrophy. Thus, elective cell-cell bond might modulate the proatherogenic activity of ATII, reducing the adverse vascular remodelling associated with AT1R activation.     

Anti-proliferative effect of rosiglitazone on angiotensin II-induced vascular smooth muscle cell proliferation is mediated by the mTOR pathway

VSMC (vascular smooth muscle cell) proliferation contributes significantly to intimal thickening in atherosclerosis, restenosis and venous bypass graft diseases. Ang II (angiotensin II) has been implicated in VSMC proliferation though the activation of multiple growth-promoting signals. Although TZDs (thiazolidinediones) can inhibit VSMC proliferation and reduce Ang II-induced fibrosis, the mechanism underlying the inhibition of VSMC proliferation and fibrosis needs elucidation. We have used primary cultured rat aortic VSMCs and specific antibodies to investigate the inhibitory mechanism of rosiglitazone on Ang II-induced VSMC proliferation. Rosiglitazone treatment significantly inhibited Ang II-induced rat aortic VSMC proliferation in a dose-dependent manner. Western blot analysis showed that rosiglitazone significantly lowered phosphorylated ERK1/2 (extracellular-signal-regulated kinase 1/2), Akt (also known as protein kinase B), mTOR (mammalian target of rapamycin), p70S6K (70 kDa S6 kinase) and 4EBP1 (eukaryotic initiation factor 4E-binding protein) levels in Ang II-treated VSMCs. In addition, PPAR-γ (peroxisome-proliferator-activated receptor γ) mRNA increased significantly and CTGF (connective tissue growth factor), Fn (fibronectin) and Col III (collagen III) levels decreased significantly. The results demonstrate that the rosiglitazone directly inhibits the pro-atherosclerotic effect of Ang II on rat aortic VSMCs. It also attenuates Ang II-induced ECM (extracellular matrix) molecules and CTGF production in rat aortic VSMCs, reducing fibrosis. Importantly, PPAR-γ activation mediates these effects, in part, through the mTOR-p70S6K and -4EBP1 system.     

EBP50 is involved in the regulation of vascular smooth muscle cell migration and cytokinesis

Ezrin, Radixin, Moesin binding phosphoprotein 50 (EBP50) is a scaffold protein that possesses two PDZ interacting domains. We have shown that, in isolated artery stimulated with noradrenaline, EBP50 interacts with several elements of the cytoskeleton. However, the contribution of EBP50 to the organization of the cytoskeleton is unknown. We have used primary cultured vascular smooth muscle cells to investigate the involvement of EBP50 in the regulation of cell architecture, motility and cell cycle, and to identify its target proteins and subsequent action mechanism. The results showed that depletion of EBP50 by siRNA transfection induced changes in cell architecture and increased cell migration. The same phenotype was induced by inhibition of myosin IIa and this effect was not additive in cells depleted for EBP50. Moreover, a larger proportion of binucleated cells was observed after EBP50 depletion, indicating a defect in cytokinesis. The identification, after co-immunoprecipitation, of a direct interaction of EBP50 with both tubulin and myosin IIa suggested that EBP50 could regulate cell migration and cytokinesis by linking myosin IIa fibers and microtubule network. Indeed, depletion of EBP50 also dismantled myosin IIa fibers and induced the formation of stable microtubules in lamellae expansions and Rac1 activation. This signaling cascade leads to the formation of lamellipodia, trailing tails and decrease of focal adhesion formation, triggering cell migration.     

Effects of statin treatment and withdrawal on angiotensin II-induced phosphorylation of p38 MAPK and ERK1/2 in cultured vascular smooth muscle cells

Abrupt discontinuation of 3-hydroxy-3-methylglutaryl-coenzyme-A-reductase inhibitors (statins) is associated with increased cardiovascular risk. To investigate the molecular mechanisms determining the increased cardiovascular risk after statin withdrawal, we studied the effects of statin treatment and withdrawal on angiotensin II (AII) actions in rat aortic vascular smooth muscle cells (VSMC) in culture. In VSMC, AII stimulated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), and of p38 mitogen-activated protein kinase (p38 MAPK), with an EC50% of 0.86 and 3 nM, respectively. Maximal stimulation was observed after 5-10 min of exposure to AII. Pretreatment with 1-3 microM simvastatin for 24h inhibited AII-mediated stimulation of ERK1/2 and p38 MAPK phosphorylation; without affecting the levels on non-phosphorylated MAPK. Washout of simvastatin produced a rebound increase above control levels of AII-mediated phosphorylation of ERK1/2 and p38 MAPK. As previously reported for other agonists, the rebound increase of AII effects was observed from 1 to 3h after statin withdrawal, and was lost at later times. The basal levels of phosphorylation and the amount of non-phosphorylated kinases were unaffected by statin withdrawal. Similar effects were observed with lovastatin. Our results suggest that statins modulate AII effects in VSMC, and that transient increases in AII effects mediated via the MAPK pathway may play a role in the vascular dysfunction associated with statin withdrawal.     

Whey peptide Isoleucine-Tryptophan inhibits expression and activity of matrix metalloproteinase-2 in rat aorta

Aortic stiffness is an independent risk factor for development of cardiovascular diseases. Activation of renin-angiotensin-aldosterone system (RAAS) including angiotensin converting enzyme (ACE) activity leads to overproduction of angiotensin II (ANGII) from its precursor angiotensin I (ANGI). ANGII leads to overexpression and activation of matrix metalloproteinase-2 (MMP2), which is critically associated with pathophysiology of aortic stiffness. We previously reported that the whey peptide Isoleucine-Tryptophan (IW) acts as a potent ACE inhibitor. Herein, we critically elucidate the mechanism of action by which IW causes inhibition of expression and activity of MMP2 in aortic tissue. Effects of IW on expression and activity of MMP2 were assessed on endothelial and smooth muscle cells (ECs and SMCs) in vitro and ex vivo (isolated rat aorta). As controls we used the pharmaceutical ACE inhibitor – captopril and the ANGII type 1 receptor blocker – losartan. In vitro, both ANGII and ANGI stimulation significantly (P < 0.01) increased expression of MMP2 assessed with western blot. Similarly, to captopril IW significantly (P < 0.05) inhibited ANGI, but not ANGII mediated increase in expression of MMP2, while losartan also blocked effects of ANGII. Signaling pathways regulating MMP2 expression in ECs and SMCs were similarly inhibited after treatment with IW or captopril. In ECs IW significantly (P < 0.05) inhibited JNK pathway, whereas in SMCs JAK2/STAT3 pathway, assessed with western blot. In vitro findings were fully consistent with results in isolated rat aorta ex vivo. Moreover, IW not only inhibited the MMP2 expression, but also its activation assessed with gelatin zymography. Our findings demonstrate that IW effectively inhibits expression and activation of MMP2 in rat aorta by decreasing local conversion of ANGI to ANGII. Thus, similar to pharmaceutical ACE inhibitor captopril the dipeptide IW may effectively inhibit ACE activity and prevent the age and hypertension associated rise of aortic stiffness.